The ex vivo production of cells engineered by material delivery through the cell wall is widely used in fundamental research and therapeutic development. DNA and RNA enable the study of the function, expression, and regulation of genes; high-throughput and patient-specific screening of gene and conventional drug therapies; therapeutic tissue engineering; and the biomanufacture of proteins and other materials. Transfection methods such as viral transduction, use of cationic and liposomal materials or polymer nanoparticles, and physical methods such as electroporation/nucleofection can have high efficiencies but poor cell viability. Inefficient cell transformation is emerging as a bottleneck for autologous and allogenic therapies. As a result, there is a significant need for an efficient, scalable, and more universal delivery method. Covaris will develop a platform for intracellular delivery based on precision sonoporation based on its Adaptive Focused Acoustics (AFATM) technology. In sonoporation, acoustic streaming generated by ultrasound-induced oscillations of gas microbubbles disturbs the cell membrane, generating transient pores for material transfer. Sonoporation has been explored with moderate success, with and without added microbubbles as cavitation nuclei. Most previous experiments were performed with poor control of parameters such as pressure amplitudes and uniformity and the proximity and size of microbubbles. Experiments which have attempted to control these parameters resulted in superior transfection rates but are not scalable. The proposed system will tightly control the acoustic field, the nature of shear-generating microbubbles, and the bubbles? proximity to cells. A highly-uniform acoustic field will be combined with a plastic microfluidic chip. Proximity to microbubbles will be controlled by either chemical means or through design of the microfluidic chip. Feedback will control acoustic intensity within the chip. Both approaches lend themselves to high-throughput transfection due to the short bubble excitation times required per transfection. Phase I will focus on exploring this concept in low throughput through different design approaches for three cell types inefficiently transfected by standard methods. The project will have three components: Development of cell culture and analysis systems using ?gold standard? chemical (lipofection) and physical (electroporation) methods; the development of an instrument and a series of embossed thermoplastic and cast elastomer microfluidic chips for precise sonoporation; and the evaluation of cross-membrane transport using fluorescent molecules (low-MW dextrans and high-MW plasmid) and transfection with a GFP-coding plasmid. The goal of the Phase I project is to increase (transfection efficiency) X (viability) by 100% with a 5-fold increase in throughput relative to pate-based methods.